1. Field of the Invention
The present invention is directed to the use of an Aspergillus oryzae protease preparation as an anti-inflammatory agent useful in the treatment of various diseases and conditions.
2. Background of the Invention
Proteolytic enzymes have been used extensively as therapeutic agents for decades. The earliest studies used pancreatic enzymes in the treatment of cancer. Later, proteolytic enzymes from non-animal sources such as the plant enzymes bromelain and papain and proteases derived from fungi such as Aspergillus sp. were investigated.
Proteases from Aspergillus oryzae are commercially used in the production of sake and soy sauce as well as in flavoring of other food products. Clinically, these enzymes have been shown to have an anti-thrombolytic/anti-hypertensive effect (Frish, E., “Clinical Review on Brinase, a Protease from Aspergillus oryzae,” Folia Haematol., 101(1):63-82 (1974), Mizuno, S., et al., “Release of Short and Proline-Rich Hypertensive Peptides from Casein Hydrolysate with an Aspergillus oryzae Protease,” J. Dairy Sci., 87:3183-3188 (2004), Sano, J., et al., “Effect of Caesin Hydrolysate Prepared with Protease Derived from Aspergillus oryzae, on Subjects with High-Normal Blood Pressure or Mild Hypertension,” J. Medicinal Food, 8(4):423-430 (2005)), anti-cancer effect (Smyth, H., et. al., “The Effects of Protease I of Aspergillus oryzae (Brinase) on Membrane Permeability and Growth of Landshutz Ascites Tumour Cells,” Int. J. Cancer, 7:476-482 (1971), U.S. Pat. No. 5,562,900), and an anti-viral effect (Knight, C., “Immunogenic Properties of PR9 Influenza Virus After Treatment with Acid Protease,” Intervirology, 14:37-43(1980), Roth, R., et al., “Proteolytic Action of Aspergillus niger Extract on Influenza Virus,” Intervirology, 14:167-172 (1980), Singh, S., et al. “Isolation, Structure, and HIV-1 Integrase Inhibitory Activity of Structurally Diverse Fungal Metabolites,” J. Ind. Microbiol. Biotechnol., 30:721-731 (2003)). In addition, proteases from Aspergillus oryzae have been shown to be potent anti-inflammatory mediators (Kolodny, A., “Double Blind Evaluation of Asperkinase, a New Proteolytic Enzyme,” Am. J. Orthopedics, 234-235 (1963), U.S. Pat. No. 6,413,512 B1, U.S. Pat. No. 3,932,618, EP 1390 542).
In many diseases and injuries there is a marked increase in circulating proinflammatory cytokine levels. This increase in cytokine expression is hypothesized to contribute to the pathology of these conditions. Infection, cancer and tissue injury can all trigger the production of cytokines, which can then enter the blood stream to alter the physiology of distant tissues, or act locally as paracrine mediators. In some diseases and injury states cytokines are beneficial to the host, but in others, cytokines are detrimental to the host. Proteases and cytokines are intimately interrelated in that cytokines are involved in regulating the production of proteases and proteases are frequently involved in the liberation of soluble cytokines, as well as in their destruction. Diseases in which cytokines play a pathological role include multiple sclerosis, type I diabetes, rheumatoid arthritis, soft tissue injury, and solid tumor malignancies. It would be beneficial therefore in these disease states to decrease the levels of circulating cytokines. This has been accomplished by treating patients with antibodies to specific cytokines, i.e. TNFα, soluble receptor antagonists, and also proteases from plant and microbial sources.
Cytokines play a major role in the manifestation of inflammation, which is a predominant biological reaction to a myriad of injurious agents and events. It is well-known that host defensive and reparative processes in inflammation can be harmful to the body's welfare. Common characteristics of inflammation are fever, swelling, bruising and pain. The body's defensive mechanisms can bring about the release of products toxic to the host or lead to destruction of its host tissues.
Detrimental consequences of inflammation include fibrin deposition, and reduction in vascularity causing changes in tissue permeability creating additional morphologic barriers to the penetration of antibodies or pharmacological agents into the injured area. Some of the autolysis products released by tissue necrosis often constitute a good medium for microorganisms and can even antagonize the antimicrobial activity of many pharmaceutical agents, thereby exacerbating the injury or infection and prolonging the recovery process.
Cytokines released in the immune response to tumor antigens, such as IL-1β and IL-6 can upregulate angiogenic factors such as vascular endothelial growth factor (VEGF) which leads to new blood vessel formation providing nutrition to the growing malignancy, thereby helping the tumor to grow.
In addition, cytokines are pathogenic mediators in many autoimmune conditions such as rheumatoid arthritis (RA), multiple sclerosis (MS) and Crohn's disease. Current treatments in RA focus on inhibiting tumor necrosis factor alpha (TNF-α) production and signaling. In animal models of MS, inhibition of interferon-γ has shown promise.
The absorption of orally-administered proteases in mammals has been extensively studied. The prevailing finding of these studies is that proteases can be partially absorbed intact, with activity preserved, from the digestive tract and subsequently distributed systemically in the blood. Proteolytic enzymes from Aspergillus oryzae are often used as digestive aids, and as such stimulate bowel movements, often times leading to diarrhea in the host.
Early in the study of proteases, it was observed that the administration of animal-derived proteases could accelerate the healing of inflamed sites. Therefore, a large database exists of clinical results from orally-administered, animal derived proteases establishing the effectiveness of these proteases as therapeutic agents for inflammatory conditions. However, a clear mechanism of physiological action for animal-derived proteases is yet to be determined. Plant proteases have also been found to have a positive effect on inflammation The largest body of evidence supporting the use of proteases for inflammatory conditions studied the effects of a mixture of papain, bromelain, trypsin, chymotrypsin, pancreatin and rutin. In most cases, the mixture was in addition to standard medical care.
It has long been established that a number of chemical compounds typically referred to as vitamins and minerals provide significant health value and treat specific medical conditions, particularly when supplied in therapeutic doses. Over the years, a number of such vitamins and minerals have been identified. For example, vitamins include A, C, D, E, and the family of B vitamins and minerals include iron, zinc, calcium and chromium. The human body does not synthesize most of these vitamins and minerals which are essential to maintaining the health of the human body. Thus these necessary vitamins and minerals must be obtained from an external source. The two most common external sources are foods and nutritional supplements. Food is typically the primary source of obtaining the necessary nutrients for maintaining health, however many people do not eat foods that consistently provide the necessary daily requirements of vitamins and minerals. Thus, vitamin and mineral nutritional supplementation has become a recognized method of meeting these daily requirements.
While certain vitamins and minerals have been shown to be essential for the maintenance an individual's health, the use of vitamin and mineral nutritional supplementation has afforded the possibility to include micro-nutrients which, although not absolutely essential to maintaining health, provide significant benefit toward maintaining health.
U.S. Pat. No. 6,413,512 B1 describes treating patients suffering from a disease resulting from increased cytokine production with a pharmaceutical composition comprising 2 or more proteases from a microbial source in an amount of between 20,000 HUT and 550,000 HUT. The protease described this patent is made using rice and/or wheat bran as the carbohydrate source.
In light of the above, the present invention is based on the surprising result that proteases from Aspergillus oryzae made using potato dextrin as the carbohydrate source are better absorbed by the proximal small intestine than proteases using rice or wheat bran as the carbohydrate source and thus are exceptionally potent anti-inflammatory mediators. Proteases made using potato dextrin as the carbohydrate source, in contrast to those made from rice and or wheat bran, reduce gastrointestinal side effects such as diarrhea. In addition, administering more than 2,000,000 HUT/day of Aspergillus oryzae protease made from potato dextrin, along with a specific multi-vitamin formulation, provides an optimal and therapeutic anti-inflammatory effect.